The invention relates to a new, selective modulatory binding site in potassium channels for screening and finding new active ingredients for the treatment of diseases, which can be attributed to a hyper excitation or a deficient excitability of neuronal cells.
Potassium channels have various definite functions in excitable cells, and not excitable cells. These functions include, for instance, the control of the membrane potential, the regulation of insulin secretion from pancreatic xcex2-cells, the control or the release of cytokines from T lymphocytes, the regulation of the salt and water equilibrium in kidney cells, and the control of the electrical excitability and synaptic plasticity of neurons. It is therefore not surprising that the activity of the potassium channels is subject to a plurality of control mechanisms, which include the redox potential of a cell, the secondary messenger systems, namely calcium and cyclo-adenosine monophosphate (c-AMP), protein kinases and phosphatases, and the membrane potential. Moreover, the structures of previously characterized potassium channel proteins have numerous variations of different basic motifs and are thus very heterogeneous. On the basis of these various functions, it is also not surprising that different potassium channels occur ubiquitously in the plant and animal kingdoms (Sewing, S., Rxc3x6per, J., and Pongs, O.: Structure and function of voltage-gated K+ channels. Euroforum 2, 21-28, 1996).
Potassium channels selectively fulfill their heterogeneous tasks due to these manifold structures and in conjunction with the very large band width of the modulation paths. Substances, which selectively modulate the potassium channels are interesting medicinal drugs for a plurality of different diseases. Potassium channels are discussed in the literature as targets for the treatment of strokes, epilepsy, Alzheimer""s disease, psychiatric diseases, sleep disorders, cardiac arrhythmiasis, diabetes type II, osmotic dysfunctions such as in the case of glaucoma, tumor cell growth, but also for inflammation processes and for learning disorders, for high blood pressure, incontinence and asthma.
It was previously possible to successfully implement the treatment of diabetes, of cardiac arrhythmiasis and of high blood pressure with selective medicinal drugs (Sewing et al., see above).
In 1998, Schrxc3x6der et al. reported for the first time that a mutation-induced, slight interference with only one member of the large family of potassium channels can substantially interfere with the fragile equilibrium between excitation and inhibition of excitable cells. In the case of this particular channel, it is a heterooligomer consisting of the subunits with the names of KCNQ2 and KCNQ3. The function of this channel is reduced by about 25% through mutation of one of the two subunits. This causes affected patients to suffer epileptic attacks already in early infancy (Schrxc3x6der, B. C., Kubisch, C., Stein, V., Jentsch, T. J., Moderate loss of function of cyclic-AMP modulated KCNQ2/KCNQ3 K+ channels causes epilepsy. Nature 396, 687-690, 1998). This channel is the first potassium channel, to which a human disease can be unambiguously assigned. Schrxc3x6der postulates that a positive modulation of this channel should produce a strong anticonvulsive effect. He concludes that, by increasing the level of the intracellular seconal messenger cAMP, the activity of the channel can be positively affected; in his in vitro systems, he was able to show that the activity of the channel actually increases as the cAMP level increases.
At about the same time, Wang et al. (Wang, H. S., Pan, Z., Shi, W., Brown, B. S., Wymore, R. S., Cohen, U. S., Dixon, J. E., McKinnon, D., KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science 282, 1890-1893, 1998) showed that the above-mentioned heterooligomeric channel, confirmed in man and consist of KCNQ2 and KCNQ3, is the molecular correlate of the M channel, the functions of which were described long ago.
The M channel is formed selectively only in neuronal cells (see Schrxc3x6der et al., and Wang et al., above) and is coupled there over intracellular signal proteins (G proteins) selectively in the central nervous system to muscarinergic sub-types of the acetylcholine receptor. The channel is not expressed in the peripheral tissue. The activity of the channel is lowered by muscarine agonists and raised by muscarine antagonists.
The muscarine agonist, pilocarpin, initiates severe convulsions in animals. The resulting destruction of the cells, in which the muscarine receptor (and also of the M channel) is expressed, causes the animal to develop spontaneous epileptic episodes after this treatment.
By means of a different muscarine agonist, oxotremorin, an essential tremor, which simulates the tremor of Parkinson""s patients, can be initiated in the animal by sub-lethal doses. Muscarine-antagonistic substances are used clinically for the treatment of this tremor.
From these presentations, which are given by way of example, it becomes clear that an indirect modulation of the M channel (initiated over muscarine receptors or caused by an increase in the cAMP level) represents a highly interesting possibility for intervening in different diseases.
In particular, this is to be shown in greater detail for several diseases.
Epilepsy
Epilepsy is characterized by the repeated occurrence of convulsions. It occurs at the rate of 0.5 to 1% of the population. Epileptic convulsions result from an abnormal synchronization and a massive discharge of a large number of nerve cells in a nerve cell association in the brain. Depending on the participation of different regions of the brain, this results in a paroxysmal temporary disturbance in motor activity (motor convulsions), emotional state, behavior or perception (Janz, D. (1985) Epilepsy: Seizures and syndromes. In: Frey, H. H. and Janz, D. eds., Antiepileptic drugs. Handbook of experimental pharmacology, Vol. 74, pp. 3-34, Springer-Verlag, Berlin, Heidelberg, New York, Tokyo); (Porter, R. J., Classification of epileptic seizures and epileptic syndromes. In: Laidlaw J., Richens A., Chadwick D., (eds.): A Textbook of Epilepsy. Churchill Livingstone, N.Y., 1993, pp. 1-22).
However, the convulsion is only one symptom, which principally can be initiated in each individual, when the stimulus for activation and synchronization is sufficiently strong. Epilepsy is therefore characterized by an increased sensitivity to external or internal stimuli for synchronization and activation. The excitability and the sensitivity of the nerve cells is greater in epileptic patients.
As mentioned above, it was possible recently to show that potassium channels, which are composed of the subunits KCNQ2 and KCNQ3, have a decisive controlling effect on the excitability of nerve cells (Schrxc3x6der et al., supra). A reduction in function of these channels by about 25% already leads to epileptic attacks in infants, who do not have adequate compensatory mechanisms at their disposal. Such a weak reduction in the function of this potassium channel was detected for a genetically determined form of epilepsy, the BFNC (benign familial neonatal convulsions) variety (Schrxc3x6der et al., supra). This potassium channel can be detected only in the brain and in nerve cells, but not in other tissue (see Schrxc3x6der et al., supra; and Wang et al., supra).
It has not yet been possible to clarify the genetic cause for other forms of epilepsy. However, the disease is always associated with an increased excitability of nerve cell networks. The current therapy tended to reduce the symptoms of the diseases. Established antiepileptic drugs, such as carbamazepine, phenytoin and lamotrigine act as use-dependent blockers of sodium channels. These channels are necessary in order to conduct cellular excitations along nerve fibers.
Sodium channel blockers reduce the conductance of excitations and in this way have an anticonvulsive action. The underlying hyper-excitation is however not reduced. Other antiepileptic drugs, such as phenobarbital, clonazepam, vigabatrin, topiramate or valproate intensify the inhibitory neurotransmission or reduce the excitory neurotransmission as they reduce the probability that an excitation will be transferred to other nerve cells. Here also, the cause of the disease, the underlying hyper-excitability of the individual nerve cells, is not affected; only the conduction of signals is reduced. However, medicinal drugs, which selectively positively modulate the above mentioned potassium channel can affect the excitability of the nerve cells directly. Since the channel can be detected only in neuronal tissue, a selective effect without side effects on other tissues is to be expected for such substances.
Alzheimer""s Disease
As already mentioned, the potassium channel, consisting of the subunits KCNQ2 and KCNQ3, represents the molecular correlate of the M channel (see Wang et al., supra). The M channel is negatively coupled over a tight coupling to a sub-type of the acetylcholine receptor, the muscarine receptor of the central nervous system. Muscarine receptors, outside of the nervous system, are not coupled with the M channel. An activation of muscarine receptors leads to a reduction in the probability that this channel is open and thus to a reduction in the function.
This is already used pharmacology. Due to a weak inhibition of acetylcholine esterase, an enzyme that decomposes acetylcholine, by means of medicinal drugs, such as Donepezil-HCl (such as is sold under the trademark Aricept(copyright)), the acetylcholine concentration in the brain is increased and the muscarine receptor activated, as a result of which the potassium channel is negatively modulated (inhibited). This results in an increase in the excitability of cholinergic nerve cells. In Alzheimer""s disease, cholinergic nerve cells are selectively destroyed, which leads to the known symptoms of loss of memory (dementia). By increasing the excitability of the remaining cholinergic nerve cells, the latter can compensate for the functions of the destroyed nerve cells, as a result of which the loss of memory is counteracted.
However, the enzyme inhibitors of the cholinesterase have decisive disadvantages. Since cholinergic signal transduction plays an important role in the whole of the body and thus also in the skeletal muscles and in other tissues, and since the substances inhibit cholinesterase everywhere, there is a plurality of side effects, such as dizziness, dyspepsia, abdominal pain, nausea and/or vomiting, diarrhea, anorexia and myalgia. Occasionally, weakness, ataxia, sleeplessness, weight loss and bradycardia also occur.
The substance, linopirdine, is in development to avoid these disadvantages. It does not affect the cholinergic system. Instead, it blocks the M channel (=KCNQ2+KCNQ3) (see Wang et al., supra). However, this medicinal drug has the disadvantage that it does not have a sufficient selectivity for the M channel. Channels with a similar affinity, which play a major role in the function of the heart muscle, are blocked. These channels are, in particular, the KCNQ1 channel, but also the eag1, erg1, erg3, elk1, Kv1.2 and Kv4.3 channels, which are widespread in the heart and in other tissue (see Wang et al., supra).
The objective of selectively inhibiting KCNQ2/3 could therefore not be achieved with medicinal drugs, which modulate the linopirdine binding site. The new ligand for the linopirdine binding site, XE991, inhibits the KCNQ1 and Kv4.3 channels with a similar affinity. These two channels are important for the functioning of the heart. A blockade of these heart-specific channels can initiate fatal arrhythmias, so that a further development of this substance is questionable.
Parkinson""s Disease
A modulation of central muscarine receptors is an aim also for the treatment of other diseases. For example, muscarine receptor antagonists are used for Parkinson""s disease to treat the symptoms, above all, the tremor. However, as with Alzheimer""s disease, the selectivity of muscarine receptor ligands for central receptors is inadequate also here, and there are undesirable side effects due to interactions with peripheral muscarine receptors and other ion channels and receptors. Since central muscarine receptors are coupled with the M channel, this function can be exercised more selectively by M channel activators than by muscarine antagonists. However, these medicinal drugs have not yet been described previously.
Neurodegenerative Diseases
Aside from the diseases mentioned above, affecting the excitability of nerve cells also plays an important role in neurodegenerative diseases (Rundfeldt, C., Potassium channels and neurodegenerative diseases. Drug News and Perspectives 12, 99-104, 1999).
Nerve cell degeneration occurs whenever there is an imbalance between energy consumption and energy supply. For example, in the case of a stroke blood is not supplied and nerve cells die. In the case of toxic hyper-excitations, as in the case of the epileptic state or amyotrophic lateral sclerosis, the cells increasingly consume energy due to hyper-excitation, and the supply of new energy is no longer adequate (see Rundfeldt et al., supra). In addition, the neurotransmitter, glutamate, is secreted by the cells. Because of the increased concentration of glutamate and the inadequate energy supply, the neurons can no longer maintain the membrane potential and depolarize. As a result of the activation of the KCNQ2/3 channel, the cells are hyper-polarized and can recover from the described noxae.
A selective lowering of the excitability of such nerve cells can therefore prevent nerve cell destruction. However, aside from lowering the temperature of the body, there was no reliable direct method of lowering the excitability of nerve cells until now (see Rundfeldt et al., supra).
Through the discovery of the significance of the KCNQ2/KCNQ3 channel for the selective control of the excitability of nerve cells, it became evident that an activation of this channel can prevent nerve cell damage. For the reasons indicated, it is necessary selectively to affect the channel with the subunits KCNQ2 and KCNQ3, to achieve a selective, for example, anticonvulsive, anti-Parkinson and neuroprotective effect upon activation and a selective memory-enhancing effect upon inhibition. Aside from the mentioned diseases, all states can be affected which are associated with a hyper-excitation or a lack of excitation of nerve cells which carry the M channel or lie in its projection fields.
This objective cannot be achieved with previously available medicinal drugs and treatment strategies.